Mixed alkanethiol self-assembled monolayers as substrates for microarraying applications

In current microarraying experiments, data quality is in large part determined by the quality of the spots that compose the microarray. Since many microarrays are made with contact printing techniques, microarray spot quality is fundamentally linked to the surface characteristics of the microarray substrate. In this work, surface coatings, consisting of self-assembled monolayers (SAMs) of mixed alkanethiol molecules, were used to control the surface properties of the microarray substrate. X-ray photoelectron spectroscopy and equilibrium contact angle measurements were performed in order to confirm the chemical content and wettability of these surface coatings. To test their performance in microarraying applications, sample microarrays were printed on these mixed alkanethiol films and then characterized with a noncontact visual metrology system and a fluorescence scanner. This work demonstrates that utilizing mixed alkanethiol SAMs as a surface coating provides spatially homogeneous surface characteristics that are reproducible across multiple microarray substrates as well as within a substrate. In addition, this paper demonstrates that these films are stable and robust as they can maintain their surface characteristics over time. Overall, it is demonstrated that SAMs of mixed alkanethiols serve as a useful surface coating, which enhances spot and therefore data quality in microarraying applications.     

Use of photolithography to encode cell adhesive domains into protein microarrays

Protein microarrays are rapidly emerging as valuable tools in creating combinatorial cell culture systems where inducers of cellular differentiation can be identified in a rapid and multiplexed fashion. In the present study, protein microarraying was combined with photoresist lithography to enable printing of extracellular matrix (ECM) protein arrays while precisely controlling "on-the-spot" cell-cell interactions. In this surface engineering approach, the micropatterned photoresist layer formed on a glass substrate served as a temporary stencil during the microarray printing, defining the micrometer-scale dimensions and the geometry of the cell-adhesion domains within the printed protein spots. After removal of the photoresist, the glass substrates contained micrometer-scale cell-adhesive regions that were encoded within 300 or 500 microm diameter protein domains. Fluorescence microscopy and atomic force microscopy (AFM) were employed to characterize protein micropatterns. When incubated with micropatterned surfaces, hepatic (HepG2) cells attached on 300 or 500 mum diameter protein spots; however, the extent of cell-cell contacts within each spot varied in accordance with dimensions of the photoresist stencil, from single cells attaching on 30 microm diameter features to multicell clusters residing on 100 or 200 microm diameter regions. Importantly, the photoresist removal process was shown to have no detrimental effects on the ability of several ECM proteins (collagens I, II, and IV and laminin) to support functional hepatic cultures. The micropatterning approach described here allows for a small cell population seeded onto a single cell culture substrate to be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. This technology will be particularly useful for high-throughput screening of biological stimuli required for tissue specification of stem cells or for maintenance of differentiated phenotype in scarce primary cells.     

Evaporative properties and pinning strength of laser-ablated, hydrophilic sites on lotus-leaf-like, nanostructured surfaces

Wetting, evaporative, and pinning strength properties of hydrophilic sites on superhydrophobic, nanostructured surfaces were examined. Understanding these properties is important for surface characterization and designing features in self-cleaning, lotus-leaf-like surfaces. Laser-ablated, hydrophilic spots between 250 mum and 2 mm in diameter were prepared on silicon nanowire (NW) superhydrophobic surfaces. For larger circumference pinning sites, initial contact angle measurements resemble the contact angle of the surface within the pinning site: 65-69 degrees . As the drop volume is increased, the contact angles approach the contact angle of the NW surface without pinning sites: 171-176 degrees . The behavior of water droplets on the pinning sites is governed by how much of the water droplet is being influenced by the superhydrophobic NW surfaces versus the hydrophilic areas. During the evaporation of sinapic acid solution, drops are pinned by the spots except for the smaller circumference sites. Pinning strengths of the hydrophilic sites are a linear function of the pinning spot circumference. Protein samples prepared and deposited on the pinning sites for analysis by matrix-assisted laser desorption ionization indicate an improvement in sensitivity from that of a standard plate analysis by a factor of 5.     

Application of partial least squares discriminant analysis and variable selection procedures: a 2D-PAGE proteomic study

2D gel electrophoresis is a tool for measuring protein regulation, involving image analysis by dedicated software (PDQuest, Melanie, etc.). Here, partial least squares discriminant analysis was applied to improve the results obtained by classic image analysis and to identify the significant spots responsible for the differences between two datasets. A human colon cancer HCT116 cell line was analyzed, treated and not treated with a new histone deacetylase inhibitor, RC307. The proteins regulated by RC307 were detected by analyzing the total lysates and nuclear proteome profiles. Some of the regulated spots were identified by tandem mass spectrometry. The preliminary data are encouraging and the protein modulation reported is consistent with the antitumoral effect of RC307 on the HCT116 cell line. Partial least squares discriminant analysis coupled with backward elimination variable selection allowed the identification of a larger number of spots than classic PDQuest analysis. Moreover, it allows the achievement of the best performances of the model in terms of prediction and provides therefore more robust and reliable results. From this point of view, the multivariate procedure applied can be considered a good alternative to standard differential analysis, also taking into account the interdependencies existing among the variables.     

Sensitive protein microarray synergistically amplified by polymer brush-enhanced immobilizations of both probe and reporter

Great challenge remains to continuously improve sensitivity of protein microarrays for broad applications. A copolymer brush is in situ synthesized on both substrate and silica nanoparticle (SNP) surface to efficiently immobilize probe and reporter protein respectively for synergistic amplification of protein microarray signals. As a demonstration, sandwich immunoassay for a cancer biomarker carcinoembryonic antigen (CEA) detection is performed on microarray platform, showing a limit of detection (LOD) of 10 pg/ml and dynamic range of 10 pg/ml to 100 ng/ml. Two orders improvement of LOD is achieved in comparison to the small crosslinker-activated substrate. The improved sensitivity is attributed to not only the high immobilization amount of both probe and reporter but also the favorite protein binding orientations offered by the flexible brushes. This work provides a universal approach to inexpensively and significantly improve protein microarray sensitivity.     

Influence of the relative humidity on aminosilane molecular grafting properties

Functional coatings with amino groups are used in a wide range of biochemistry-related applications. A technological platform that takes advantage of the affinity between amino-functionalized coatings and biomolecules is the DNA microarray methodology. Reliability and reproducibility of the microarray data strongly depend on the quality of the substrate; therefore, a proper awareness of how the storage conditions affect the amino-functionalized coatings is necessary. In this work we have studied the influence of different relative humidity levels on amino-methyl-silane coatings prepared via sol–gel methodology. Drops of a buffer solution containing a luminescent dye have been deposited (or spotted) on the coating; the dye molecules react with the amino-groups and leave circular luminescent marks (spots) on the substrate surface. Shape and luminescence of the spots, as well as background signals, have been monitored using a microarray laser scanner. With the proposed protocol we have measured the changes of these variables due to different storage conditions. FT-IR measurements have been performed to investigate the related chemistry changes.     

Allergen microarrays on high-sensitivity silicon slides

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.     

Transport at the air/water interface is the reason for rings in protein microarrays

Fabricating a protein microarray involves the deposition of nanoliter droplets of solutions on a solid surface. Instead of uniform spots, one often observes ring-like structures that add to the difficulty in quantification. We show that the accumulation of proteins at the air/water interface of the nanodroplet is the reason. Transformation to a uniform spot can be achieved via the addition of competitive surfactants or the control of surface reaction kinetics.     

Surface characterization of carbohydrate microarrays

Carbohydrate microarrays are essential tools to determine the biological function of glycans. Here, we analyze a glycan array by time-of-flight secondary ion mass spectrometry (ToF-SIMS) to gain a better understanding of the physicochemical properties of the individual spots and to improve carbohydrate microarray quality. The carbohydrate microarray is prepared by piezo printing of thiol-terminated sugars onto a maleimide functionalized glass slide. The hyperspectral ToF-SIMS imaging data are analyzed by multivariate curve resolution (MCR) to discern secondary ions from regions of the array containing saccharide, linker, salts from the printing buffer, and the background linker chemistry. Analysis of secondary ions from the linker common to all of the sugar molecules employed reveals a relatively uniform distribution of the sugars within the spots formed from solutions with saccharide concentration of 0.4 mM and less, whereas a doughnut shape is often formed at higher-concentration solutions. A detailed analysis of individual spots reveals that in the larger spots the phosphate buffered saline (PBS) salts are heterogeneously distributed, apparently resulting in saccharide concentrated at the rim of the spots. A model of spot formation from the evaporating sessile drop is proposed to explain these observations. Saccharide spot diameters increase with saccharide concentration due to a reduction in surface tension of the saccharide solution compared to PBS. The multivariate analytical partial least squares (PLS) technique identifies ions from the sugars that in the complex ToF-SIMS spectra correlate with the binding of galectin proteins.     

Development of a biologically relevant calcium phosphate substrate for sum frequency generation vibrational spectroscopy

A novel biologically relevant composite substrate has been prepared consisting of a calcium phosphate (CaP) layer formed by magnetron sputter-coating from a hydroxyapatite (HA) target onto a gold-coated silicon substrate. The CaP layer is intended to mimic tooth and bone surfaces and allows polymers used in oral care to be deposited in a procedure analogous to that used for dental surfaces. The polymer cetyl dimethicone copolyol (CDC) was deposited onto the CaP surface of the substrate by Langmuir Blodgett deposition, and the structure of the adsorbed layer was investigated by the surface specific technique of sum frequency generation (SFG) vibrational spectroscopy. The gold sublayer provides enhancement of the SFG signal arising from the polymer but plays no part in the adsorption of the polymer. The surface morphology of the substrate was investigated using SEM and AFM. The surface roughness was commensurate with that of the thermally evaporated gold sublayer and uniform over areas of at least 36 mum(2). The chemical composition of the CaP-coated surface was determined by FTIR and TOF-SIMS. It was concluded that the surface is primarily calcium phosphate present as a mixture of amorphous, non-hydroxylated phases rather than solely stoichiometric hydroxyapatite. The SFG spectra from CDC on CaP were closely similar, both in resonance wavenumbers and in their relative intensities, with spectra of thin films of CDC recorded directly on gold. Application of previous analysis of the spectra of CDC on gold therefore enabled interpretation of the polymer orientation and conformation on the CaP substrate.     

Observation of RHEED intensity variations during ALE-Growth of CdTe-Epilayers

We present the first reflected-high-energy electron diffraction (RHEED) observations during atomic layer epitaxy (ALE) growth of CdTe on GaAs substrates. The evolution of the RHEED pattern in the initial growth stages shows that, regardless of the large lattice mismatch, growth becomes two-dimensional after the deposition of a few monolayers. We have observed intensity variations of two RHEED spots under surface resonance conditions which make the spots sensitive to either Cd- or Te-deposition. We show that this new approach is superior to the observation of the specular spot for the measurement of surface coverages and adsorption kinetics. The Cd- and Tespots have been monitored during CdTe-ALE. The observed intensity variations can be explained by simple adsorption models. From the change in the spot intensities with substrate temperature during a permanent Cd- or Teexposure of the sample we deduce a drop in the Cd- and Te-surface coverage from 1 to 0.5 at substrate temperatures higher than 315 °C.     

Disposable Amperometric Immunosensor for the Determination of the E‐Cadherin Tumor Suppressor Protein in Cancer Cells and Human Tissues

This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H₂O₂/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL^?1) and a detection limit as low as 0.16 ng mL^?1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL^?1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated.     

Formation and characterization of DNA microarrays at silicon nitride substrates

A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.     

n-型多孔Si/TiO2纳米线异质结构的制备及光电化学性能

采用光辅助电化学腐蚀法制备了n-型多孔硅衬底, 再采用水热法在其表面生长TiO₂纳米线制得了三维n-型多孔Si/TiO₂纳米线异质结构. 通过X射线衍射、 扫描电子显微镜和X射线能量散射等表征证实了n-型多孔Si/TiO₂纳米线异质结构的形成. 紫外-可见漫反射光谱测试结果表明, n-型多孔硅与TiO₂纳米线的复合提高了紫外-可见波段的光吸收. 光电性能测试结果表明, 3个样品中n-型多孔Si/TiO₂纳米线异质结作为光电极的光电流最高, 这说明n-型多孔Si/TiO₂纳米线作为光电极具有更高的光电化学分解水性能.     

A porous silicon immunoassay platform for fluorometric determination of α-synuclein in human cerebrospinal fluid

Levels of total and/or oligomeric α-synuclein may be used as a biomarker tool to aid in the diagnosis and development of new disease-modifying therapies. We report here on a porous silicon antibody microarray for the fluorimetric determination of cerebrospinal fluid levels of total α-synuclein, a protein involved the pathology of Parkinson’s disease. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, and this offers a large binding capacity for capturing probe molecules. Porous silicon also warrants efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. The platform requires 10 μL of cerebrospinal fluid, and each test requires 4 h for assay only (including immobilization of capturing antibody). The limit of detection is 35 pg mL^?1 of α-synuclein in cerebrospinal fluid, and the dynamic analytical range extends from 0.01 to 100 ng·mL^?1.

Improved silicon nitride surfaces for next-generation microarrays

This work reports how the use of a standard integrated circuit (IC) fabrication process can improve the potential of silicon nitride layers as substrates for microarray technology. It has been shown that chemical mechanical polishing (CMP) substantially improves the fluorescent intensity of positive control gene and test gene microarray spots on both low-pressure chemical vapor deposition (LPCVD) and plasma-enhanced chemical vapor deposition (PECVD) silicon nitride films, while maintaining a low fluorescent background. This results in the improved discrimination of low expressing genes. The results for the PECVD silicon nitride, which has been previously reported as unsuitable for microarray spotting, are particularly significant for future devices that hope to incorporate microelectronic control and analysis circuitry, due to the film's use as a final passivating layer.     

Link test--A statistical method for finding prostate cancer biomarkers

We present a new method, link-test, to select prostate cancer biomarkers from SELDI mass spectrometry and microarray data sets. Biomarkers selected by link-test are supported by data sets from both mRNA and protein levels, and therefore results in improved robustness. Link-test determines the level of significance of the association between a microarray marker and a specific mass spectrum marker by constructing background mass spectra distributions estimated by all human protein sequences in the SWISS-PROT database. The data set consist of both microarray and mass spectrometry data from prostate cancer patients and healthy controls. A list of statistically justified prostate cancer biomarkers is reported by link-test. Cross-validation results show high prediction accuracy using the identified biomarker panel. We also employ a text-mining approach with OMIM database to validate the cancer biomarkers. The study with link-test represents one of the first cross-platform studies of cancer biomarkers.     

Dendrimer-mediated transfer printing of DNA and RNA microarrays

This paper describes a new method to replicate DNA and RNA microarrays. The technique, which facilitates positioning of DNA and RNA with submicron edge resolution by microcontact printing (muCP), is based on the modification of poly(dimethylsiloxane) (PDMS) stamps with dendrimers ("dendri-stamps"). The modification of PDMS stamps with generation 5 poly(propylene imine) dendrimers (G5-PPI) gives a high density of positive charge on the stamp surface that can attract negatively charged oligonucleotides in a "layer-by-layer" arrangement. DNA as well as RNA is transfer printed from the stamp to a target surface. Imine chemistry is applied to immobilize amino-modified DNA and RNA molecules to an aldehyde-terminated substrate. The labile imine bond is reduced to a stable secondary amine bond, forming a robust connection between the polynucleotide strand and the solid support. Microcontact printed oligonucleotides are distributed homogeneously within the patterned area and available for hybridization. By using a robotic spotting system, an array of hundreds of oligonucleotide spots is deposited on the surface of a flat, dendrimer-modified stamp that is subsequently used for repeated replication of the entire microarray by microcontact printing. The printed microarrays are characterized by homogeneous probe density and regular spot morphology.     

Protein arrays on high-surface-area plasma-nanotextured poly(dimethylsiloxane)-coated glass slides

Treatment of poly(dimethylsiloxane) (PDMS) surfaces with SF(6) plasma results in the creation of high-surface-area nanotextured surfaces that considerably favour protein adsorption with respect to untreated ones. In order to employ such nanotextured surfaces as substrates for microarrays to be created and analysed using standard instrumentation, we fabricated thin PDMS films on top of standard low-cost microscope glass slides. The properties of both untreated and plasma-treated PDMS-coated slides towards spotting of protein solutions were evaluated in terms of spot signal intensity and homogeneity as well as of spot shape and size. It was found that the plasma-treated PDMS-coated glass slides provided highly homogeneous spots (mean intra-spot variation 7.6%) with spot signal intensity 6-times higher than that obtained using the untreated ones. In addition, comparison with commercially available polystyrene and aminosilanized-glass microarray slides showed that the proposed slides provided 3-times higher spot signal intensity and 2-times lower intra-spot signal variation. In addition, the implementation of long-aged-after-plasma-treatment nanotextured PDMS-coated glass slides provided spots whose shape and size matched those of the spotting tip. As a consequence, denser arrays of variable spot shape can be created using SF(6) plasma-treated PDMS-coated slides instead of standard microarray slides opening new potentials for bioanalytical applications.     

超亲疏水图案改善阵列芯片质量

本文提出在超疏水表面加工超亲水圆点图案为阵列基底制作免疫蛋白微阵列, 从而减轻“咖啡环效应”, 改善阵列芯片质量.